http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, 2014; 44(8): 708–715 ! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2014.895880
RESEARCH ARTICLE
Acyclic nucleoside phosphonates: a study on cytochrome P450 gene expression Jana Nekvindova1, Juan Antonio Contreras2, Peter Juvan2,3, Klementina Fon Tacer2,4, Pavel Anzenbacher1, Zdenek Zidek5, Michaela Kopecna Zapletalova1, Damjana Rozman2, and Eva Anzenbacherova1 1
Faculty of Medicine and Dentistry, Institute for Molecular and Translational Medicine, Palacky University, Olomouc, Czech Republic, Faculty of Medicine, Centre for Functional Genomics and Biochips, Institute of Biochemistry, Ljubljana, Slovenia, 3Artificial Intelligence Laboratory, Faculty of Computer and Information Science, University of Ljubljana, Ljubljana, Slovenia, 4Physiology Department, Southwestern Dallas Medical Center, University of Texas, Dallas, TX, USA, and 5Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
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2
Abstract
Keywords
1. Nucleotide analogues comprise an important class of drugs used in treatment of viral infections but also cancer. These drugs affect the structural integrity of DNA and activate different pathways and processes in the cell and may directly or indirectly influence the drug metabolizing system. Adefovir dipivoxil (AD) and tenofovir disoproxil (TD) are nucleotide analogues approved for the treatment of chronic hepatitis B and/or HIV/AIDS infection. 2. To evaluate the risk of their drug–drug interactions on the level of drug metabolism, an effect of both compounds on cytochromes P450 expression was studied using cDNA microarrays, real-time RT-PCR and immunoblotting. Mice were given intraperitoneally 25 mg/kg of AD or TD, respectively. As a positive control, a combination of prototypic cytochromes P450 (CYP) inducers, phenobarbital and b-naphthoflavone was chosen. 3. The data obtained showed a significant CYP induction in the positive control group, but no clinically significant induction of CYP genes by AD or TD was observed. Our results support the evidence of safety of AD and TD with respect to drug–drug interactions based on enzyme induction. These findings are important as a plethora of new antivirals of different types are being tested and introduced to clinical practice, mostly to be used in combinations.
Adefovir, antiviral, CYP, drug metabolism, induction, PMEA, PMPA, tenofovir
Introduction Drug side effects or toxicity as well as failure of therapy are often caused by drug–drug interactions on the level of enzymes of drug metabolism. Here, enzyme induction or inhibition leading to changes in metabolic activity of an enzyme may result in changed levels of the drugs metabolized. In particular cases, this includes also an under dosing of drugs that need to be enzymatically activated due to enzyme deficiency or, on the other hand, an ineffective detoxication and elimination of drugs due to inhibition of enzymes responsible for their inactivation. Most of the drug interactions are rather unpredictable without a systematic experimental and clinical testing. Especially for drugs that directly interact with DNA synthesis and reparation and/or
Address for correspondence: Jana Nekvindova, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 5, 779 00 Olomouc, Czech Republic. Tel: +420 495 832 767. E-mail: [email protected] Eva Anzenbacherova, Institute of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic. Tel: +420 585 632 321. E-mail: [email protected]
History Received 8 January 2014 Revised 13 February 2014 Accepted 14 February 2014 Published online 5 March 2014
may affect cell cycle and/or different cell signalling pathways, an effect on transcription of various genes should be carefully evaluated. A solid base for such an evaluation of a particular drug in terms of its drug interactions is provided by in vitro and in vivo testing of the drug’s effect on the cytochrome P450 system. Cytochromes P450 (CYP, pl. CYPs) are the most important drug metabolizing enzymes in man (Anzenbacher & Anzenbacherova´, 2001; Guengerich, 1995). They also participate in synthesis of important endogenous compounds, e.g. steroid hormones, prostaglandins and thromboxanes (Anzenbacher & Anzenbacherova´, 2001). Regulation of their expression and activity is a multi-level complex process that is despite all current knowledge in this field not completely understood. Acyclic nucleoside phosphonates are modern antivirals with a potent activity against many DNA viruses and retroviruses. Their value consists in a broader spectrum of activity, a longer duration of their effect and a lower risk of emerging viral resistance to the therapy (De Clercq & Holy, 2005). Chemical structure of the drugs is depicted in Figure 1. Adefovir dipivoxil (AD) (bis(pivaloyloxymethyl)ester of 9-[(2-phosphonomethoxy) ethyl]adenine) is a nucleoside
Acyclic nucleoside antivirals and P450 expression
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Figure 1. Structure of the compounds studied.
analogue approved for treatment of chronic hepatitis B, where only seven drugs are currently available: entecavir (Baraclude), lamivudine (Epivir-HBV), AD (Hepsera), interferon alpha-2b (Intron A), pegylated interferon (Pegasys), telbivudine (Tyzeka) and tenofovir (Viread) (FDA, 2011). It can be used also for the treatment of lamivudine-resistant HBV mutants (Benhamou, 2006; Nunez & Soriano, 2005; Zhang et al., 2006). For hepatitis B suppression, AD is given at a dose of 10 mg daily. Higher doses (60 or 120 mg/day) suppress also HIV viral replication but these are nephrotoxic in man. Tenofovir disoproxil (TD) (bis-isopropyloxycarbonyloxymethyl ester of 9-R-[2-(phosphonomethoxy)propyl]adenine) is used – as its fumarate (tenofovir DF, VireadÕ ) – in antiretroviral therapy (ART) of an HIV/AIDS infection; currently, it is approved also for the treatment of chronic hepatitis B (Delaney et al., 2006; FDA, 2011). For HIV infection, tenofovir DF is included in first line therapeutic regimen according to the June 2013 version of the consolidated guidelines on general HIV care and the use of antiretroviral drugs for treating and preventing HIV infection (WHO, 2013a). Recently, several combinations of tenofovir DF with other antivirals have been approved by FDA as a single preparation: Truvada (tenofovir DF + emtricitabine), Atripla (tenofovir DF + emtricitabine +efavirenz), Complera (emtricitabine + rilpivirine hydrochloride + tenofovir DF) and four combination Stribild (cobicistat + elvitegravir + emtricitabine + tenofovir DF) but more and new combinations are clinically tested and standardized. The need for safe drugs becomes more important both medically and socioeconomically with the rapidly increasing number of drugs introduced to the market and improving of the peoples’ access to pharmacotherapy. According to the World Health Organization (WHO) data, there were 35.3 million people suffering from HIV/AIDS infection. More than 9.7 million were receiving ART in 2012. Of this, 630 000 were children. This is over 30-fold increase in 10 years and close to a 20% increase in just 1 year (WHO, 2013b) For hepatitis B, there are 240 million of chronic cases and 600 000 people are estimated to die every year because of HBV infection (WHO, 2013c). Since both adefovir and tenofovir are highly important in the treatment of HBV and HIV infections, as well as they are used in combinations, the studies aiming at evaluation of their potential of drug– drug interactions are of particular interest. According to prescribing information, tenofovir DF interacts with didanosine (mechanism unknown) and HIV-1 protease inhibitors,
e.g. atazanavir and ritonavir (increased absorption of tenofovir DF). Concerning the possibility of drug interactions on the level of cytochromes P450, an inhibition of activities several human microsomal CYP forms by AD, TD and their active forms, adefovir and tenofovir, has been observed (Nekvindova´ et al., 2006). The most significant inhibition occurred in the case of CYP2C9 and CYP3A4; tenofovir and TD inhibited also CYP2E1 and CYP1A2 activities. These findings were in contrast to previously published data stating that the drugs are not substrates, inhibitors or inducers of CYPs (Kearney et al., 2000, 2004). However, as the inhibition occurred in rather high concentrations of these drugs, the inhibition of CYP activities is most probably not clinically significant. This sequential study is focused on evaluation of another mechanism of drug interactions, namely, on the potential of AD and TD to induce cytochromes P450 which has not been evaluated to date.
Materials and methods Animals and RNA preparation Animal experiments were evaluated and approved by Ethical committee of the Palacky University, Faculty of Medicine and Dentistry. Female mice of the inbred strain C57BL/6, 8–10 weeks old, were purchased from Charles River Deutschland (Sulzfeld, Germany). They were kept in transparent plastic cages and maintained in an Independent Environmental Air Flow Animal Cabinet (ESI Flufrance, Wissous, France). Lighting was set from 6 AM to 6 PM, temperature set at 22 C. Animals were randomly separated into four groups and treated for three consecutive days by intraperitoneal injections as following: AD and TD groups (five mice in each group) were given 25 mg/kg of AD or TD, respectively. PC (positive control) group of four animals was treated with 60 mg/kg phenobarbital plus one dose of 80 mg/kg of b-naphthoflavone on day two. Five animals of NC (negative control) group were injected 200 ml of 0.9% saline solution. On day four, animals were killed by cervical dislocation. Total RNA was isolated from liver tissue samples stored in RNAlater (Qiagen, Hilden, Germany) using TRI REAGENT (MRC, Cincinnati, OH). RNA concentration and purity was measured by spectrophotometry at 260, 280 and 230 nm using an Infinite M200 micro plate reader (Tecan, Ma¨nnedorf, Switzerland). RNA integrity was determined on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano LabChip kit (both Agilent Technologies, Santa Clara, CA).
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Table 1. Primer sequences for real-time PCR. Cyp1a2 Cyp2a4 Cyp2d9 Cyp2e1 Cyp3a13 Cyp8b1 Cyp2b10 Cyp51 LDLr 18S rRNA
50 -GCACTACCAAGACTTCAACAAGAACA-30 50 -AGGTACAGAAGAAACAGAGGACACTTC-30 50 -CCAACCCCATGCTGAACAAATCCAC-30 50 -GTTCCAGGAGTACAAGAACAAGGGG-30 50 -TGGGGACGATTCTTGCTTACCAGAA-30 50 -AAGGCTGGCTTCCTGAGCTT-30 50 -CAATGTTTAGTGGAGGAACTGCG-30 50 -ACGCTGCCTGGCTATTGC-30 50 -AGGCTGTGGGCTCCATAGG-30 50 -CGCCGCTAGAGGTGAAATTC-30
50 -TGTGGTGACTGTGTCAAAGCCAG-30 50 -TATTATTCCTATTGACAACATAGTATAATTTCCCC-30 50 -GGATACGCAAGAGTATCGGGAATGC-30 50 -CCATTCCCCAGTCACGGAGGAT-30 50 -GCTGTCGACCGTCATACAACCCC-30 50 -AACAGCTCATCGGCCTCATC-30 50 -CACTGGAAGAGGAACGTGGG-30 50 -TTGATCTCTCGATGGGCTCTATC-30 50 -TGCGGTCCAGGGTCATCT-30 50 -TTGGCAAATGCTTTCGCTC-30
Gnerre et al. (2005); Yang et al. (2001) (Cyp51, LDLr); Gutala & Reddy (2004) (18S rRNA).
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Microarray hybridization cDNA microarrays were developed in Centre for Functional Genomics and Bio-chips (CFGBC) of the University of Ljubljana. The arrays contained probes for 151 genes involved in cholesterol metabolism including genes of 40 cytochrome P450 forms (Cyp1a1, 1a2, 1b1, 2a4, 2a12, 2b9, 2b10, 2b13, 2c40, 2d22, 2e1, 2f2, 2g1, 2j5, 2j6, 3a11, 3a13, 3a25, 3a41, 4b1, 4f14, 5a1, 7a1, 7b1, 8a1, 8b1, 11a1, 11b2, 17a1, 19a1, 20a1, 21a1, 24a1, 26a1, 26b1, 27a1, 27b1, 39a1, 46a1 and 51a1). The hybridization protocol was similar to previously published (Rezen et al., 2008, 2009). Briefly, indirect amino-allyl (aa-) labelling and hybridization protocol was used. cDNA synthesis and incorporation of aa-dUTP by reverse transcription was performed using SuperScriptTM III Reverse Transcriptase (Invitrogen, Carlsbad, CA) with oligo dT primers. Luciferase Control RNA (Promega, Fitchburg, WI) and Lucidea Universal ScoreCardTM RNA (test and reference RNA mixes) were used as internal controls and calibrators. Cy3 and Cy5 NHS esters were coupled to aacDNA, purified samples were hybridized overnight (16 h) on the microarrays. When hybridization was done, array slides were washed and scanned with Tecan LS200 Scanner (Tecan, Ma¨nnedorf, Switzerland). Fluorescence images were obtained and analyzed using Array-Pro Analyzer software (Media Cybernetics, Rockville, MD). Data normalization and analysis were performed using Orange (Curk et al., 2005; Demsar et al., 2004) software. Direct comparison design was used in all microarray experiments. All the samples were hybridized against pooled negative control samples and a dye-swap was used to produce two technical replicates. Statistical significance evaluation of the normalized Cy3 and Cy5 fluorescence was done by Student’s t-test at levels 0.05 and 0.1 for a more complex evaluation. Real-time RT-PCR First strand cDNAs were synthesized from total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers (Promega). RNA was pre-treated with DNase I (Sigma-Aldrich, St. Louis, MO) and protected with RNase OUT (Invitrogen). Real-time PCR was performed on ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Target genes were amplified using the following set of primers listed in Table 1. 18S rRNA was used as an internal control for other genes data normalization. Optimized PCR reactions were performed in 20 ml volume in 96well plate format, using hot-start technology Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) with ROX
reference dye. Carry-over contamination was controlled by using non-template control samples. Thermal profile of the cycling program was: 50 C for 2 min hold (UDG incubation), 95 C for 2 min hold (Taq polymerase denaturation/activation) and up to 40 cycles of 95 C for 15 s and 60 C for 60 s). Data analysis was performed on ABI 7900HT software and statistic evaluation was done in Microsoft Office Excel 2003 using ddCT calculation method (Pfaffl, 2001) comparing each sample from drug-treated animals to each of the negative control animals (Table 1).
Results First, the microarray experiment was performed to get a global view over expression changes. Then a set of genes was chosen for a detailed examination by real-time RT-PCR and where a specific antibody was available, immunoblotting was done to confirm the results on the protein level. The data are presented according to this sequence and individual groups treated by different drugs are compared. All the results obtained were in concordance and gave a confident idea about the CYP inductions by the drugs used in the experiment. Microarray experiment Raw microarray data and experimental design details have been deposited to Gene Expression Omnibus database (GEO, http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE47698. Samples from drug-treated animals were hybridized one by one against pooled negative control samples. Statistical analysis of normalized Cy5 and Cy3 fluorescence signals of individual probes on the array revealed a set of genes differentially expressed in all groups of drug-treated animals when compared to negative controls treated by vehiculum alone. The outcoming set of genes was the largest in the positive control group (phenobarbital plus b-naphthoflavone treated), fewer genes were differentially expressed in animals treated by TD and only minor changes were detected in animals treated by AD (Table 1). Log2 ratio was evaluated and a biological cut off was set to ±0.9 (somewhat 52-fold up- or down-regulation) according to statistical significance, experimental design and mapping of known effects of CYP inducers. Major changes were detected in the positive control (PC) group and target genes, which were Cyp1a2 for b-naphthoflavone and Cyp2b10 for phenobarbital. In this PC group, an up-regulation of 12-fold (log2 ratio 3.584) was observed for Cyp1a2 and nearly 5-fold (log2 ratio 4.8) for Cyp2b10 as it is depicted in Table 2.
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Table 2. Differentially expressed genes in control- and drug-treated groups, statistically significant changes at p ¼ 0.05.
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Gene symbol
Gene name
Positive control (phenobarbital plus b-naphtoflavone) Cyp1a2 Cytochrome P450 1a2 Cyp2b10 Cytochrome P450 2b10 Cyp2b13 Cytochrome P450 2b13 Abcc3 ATP-binding cassette, sub-family C (CFTR/MRP), member 3 Cyp3a41 Cytochrome P450 3a41 Cyp3a13 Cytochrome P450 3a13 Cyp51a1 Cytochrome P450 51a1 Dhcr24 24-Dehydrocholesterol reductase Cyp2a4 Cytochrome P450 2a4 Nsdhl AD(P) dependent steroid dehydrogenase-like Lss Lanosterol synthase Fdps Farnesyl diphosphate synthetase Actb2 Actin, beta Cyp8b1 Cytochrome P450 8b1 Cyp2a12 Cytochrome P450 2a12 Lip1 Lysosomal acid lipase A Alas1 Aminolevulinic acid synthase 1 Ppia Peptidylprolyl isomerase A Acss2 Acyl-CoA synthetase short-chain family member 2 Apoa1 Apolipoprotein A-I Cyp2c40 Cytochrome P450 2c40 Cyp2f2 Cytochrome P450 2f2 Abcc2 ATP-binding cassette, sub-family C (CFTR/MRP), member 2 Ppara Peroxisome proliferator activated receptor alpha Cyp3a25 Cytochrome P450 3a25 Slc10a1 Solute carrier family 10 (sodium/bile acid cotransporter family), member 1 Nr1d2 Nuclear receptor subfamily 1, group D, member 2 Adefovir Scap SREBP cleavage activating protein (SREBP chaperone) Cyp26a1 Cytochrome P450 26a1 Tenofovir Ebp Phenylalkylamine Ca2+ antagonist (emopamil) binding protein Scarb1 Scavenger receptor class B, member 1 Acss2 Acyl-CoA synthetase short-chain family member 2 Cyp26a1 Cytochrome P450 26a1 Rxrb Retinoid X receptor beta Abcb4 ATP-binding cassette, sub-family B, member 4 (P-gp 2, MDR2) Gapd2 Glyceraldehyde-3-phosphate dehydrogenase Sc4mol Sterol-C4-methyl oxidase-like Ldlr Low-density lipoprotein receptor Ncor1 Nuclear receptor co-repressor 1, RXR interacting protein 13 Nr2b1 Retinoid X receptor alpha Cyp21a1 Cytochrome P450 21a1 Cyp51a1 Cytochrome P450 51a1 Mvk Mevalonate kinase Hmgcs1 3-Hydroxy-3-methylglutaryl-Coenzyme A synthase 1 Dhcr7 7-Dehydrocholesterol reductase
Log2 ratio
Fold change
3.584 2.258 1.881 1.836 1.641 1.585 1.336 1.273 1.211 1.205 1.135* 1.057 0.996 0.952 0.924 0.919 0.865 0.792 0.79 0.786 0.689 0.622 0.574 0.166 0.675* 0.418* 0.293*
12.0 4.8 3.7 3.6 3.1 3.0 2.5 2.4 2.3 2.3 2.2 2.1 2.0 1.9 1.9 1.9 0.5 1.7 0.6 1.7 0.6 0.6 1.5 0.9 1.6 0.7 0.8
0.376 0.536*
1.3 0.7
0.952 0.845 0.844 0.838 0.827 0.815 0.763 0.748 0.568 0.528 0.304 0.728* 0.569* 0.494* 0.414* 0.346*
1.9 1.8 0.6 0.6 1.8 1.8 1.7 0.6 1.5 1.4 1.2 1.7 0.7 1.4 1.3 1.3
Biological significance cut-off was set to an expression change of log2ratio ±0.9 (2-fold up- or down-regulation). Other statistically significant differences are shown for better illustration. *p ¼ 0.1.
Real-time RT-PCR The microarray results were confirmed by quantitative PCR where appropriate and a high concordance of both methods was seen. Genes with a suggested key role in drug metabolism in mice and genes that showed significant changes in the microarray experiment were selected for real-time PCR assays, namely Cyp1a2, Cyp2a4, Cyp2d9, Cyp2e1, Cyp3a13, Cyp8b1, Cyp2b10, Cyp51 and LDLr. Relative expression (RE) of a gene of interest was calculated by ddCT method against a selected reference gene (18S rRNA), always as an average of three technical replicates. At first, RE values (RE) ¼ 2 ddCT of all drugtreated samples compared to negative control samples were expressed by the logic ‘‘each to each’’, then an average value
for a particular sample (drug-treated animal) was calculated and finally an average RE with its standard deviation and standard error of the mean was obtained for the individual groups (AD, TD, PC, NC) (Table 3). Comparison of the positive results, where a gene expression change was detected by the microarray as well as by realtime PCR is necessary as a validation of the microarray results. Similar trends as well as value ranges were observed for most of the results, where a difference was seen the realtime results is considered to be correct as it is more specific, sensitive and accurate. Nevertheless, all but one significant gene expression changes were confirmed. The only exception was LDLr gene in TD group, where the discrepancy was related to the very low RE change (log2 ratio was close to 0), in other words, the gene expression was almost the same
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Table 3. Relative expression of selected genes – real-time PCR results. AD
TD
PC
NC
Gene
RE
SD
SEM
RE
SD
SEM
RE
SD
SEM
RE
SD
SEM
Cyp1a2 Cyp2a4 Cyp2d9 Cyp2e1 Cyp3a13 Cyp8b1 Cyp2b10 Cyp51 LDLr Cyp26a1
0.49 2.02 0.72 0.67 0.8 1.31 0.75 0.94 0.78 1.02
0.17 1.57 0.51 0.22 0.16 1.11 0.24 0.59 0.21 1.19
0.13 1.1 0.42 0.19 0.14 0.79 0.19 0.51 0.17 1.76
0.68 1.81 0.81 0.84 0.88 0.68 0.72 0.53 0.78 0.75
0.21 1.42 0.4 0.26 0.22 0.57 0.22 0.26 0.25 1.18
0.16 0.99 0.34 0.22 0.18 0.43 0.17 0.2 0.21 0.85
8.53 1.63 2.47 0.54 2.09 1.58 26.36 2.18 0.72 1.32
2.8 1.38 1.03 0.27 0.48 0.72 9.72 0.92 0.24 1.97
2.23 0.98 0.86 0.24 0.38 0.56 7.77 0.74 0.19 1.48
1.05 1.37 1.24 1.12 1.02 1.16 1.04 1.04 1.03 4.83
0.32 1.21 0.87 0.55 0.22 0.66 0.28 0.28 0.26 9.87
0.25 0.85 0.63 0.41 0.18 0.5 0.22 0.22 0.18 6.11
AD, adefovir dipivoxil; TD, tenofovir disoproxil; PC, positive control (phenobarbital + b-naphthoflavone); NC, negative control group; RE, relative expression (average fold induction, 1.00 ¼ 100% RENC); SD, standard deviation; SEM, standard error of the mean. Gene expression variability of the target genes in the negative control group is shown for illustration.
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was limited and where the antibody was targeted against human isoform, poor cross-reactivity with murine substrates led to an ineffective binding of the antibody. Reproducible results were obtained for CYP1A2 where a massive enzyme induction was detected in the positive control group and even specific degradation products of this protein were present on the blot (Figure 3). Samples in other groups were similar to each other as well as to negative control samples, i.e. there was no significant Cyp1a2 gene expression change on the protein level in AD or TD group. Incubation of the membranes with an anti-CYP51 antibody gave repeatedly similar results even if the reaction was not optimal and the signal intensity always varied between individual membranes. Using internal standards (negative/positive group samples), the comparison was possible. The anti-CYP26A1 was antihuman and despite all optimization effort it never reacted to murine samples (positive reaction is seen only for human liver microsomes, Figure 3).
Discussion
Figure 2. A comparison of the relative expression results obtained by cDNA microarrays Sterolgene v.1 Mouse and real-time RT-PCR. TD, tenofovir disoproxil-treated group; PC, positive control group; RE, relative expression.
in drug-treated animals as in negative controls. Expression of other genes was confirmed by real-time PCR even despite a different experimental approach and a completely different statistical analysis which increases the overall confidence of the results. The genes with highest correlation were Cyp1a2 and Cyp2b10 (induced by phenobarbital and b-naphthoflavone), Cyp2a4, 3a13, 8b1 and Cyp51 in the positive control group and Cyp51 gene (down-regulation) in the TD group as depicted in Figure 2. Western blotting was used to visualize gene induction on the protein level. The spectrum of antibodies on the market
Adefovir and tenofovir are potent antivirals successfully used in pharmacotherapy of chronic hepatitis B and HIV/AIDS infection, respectively. They are active against a broad spectrum of DNA viruses, e.g. polyoma-, papilloma-, adeno-, herpes- and poxviruses (De Clercq et al., 2005). The producer states on the drug’s web sites (http:// www.hepsera.com, http://www.viread.com) that these drugs are not substrates of CYPs and even in 4000 (adefovir) or 300 (tenofovir) higher concentrations than those reached in vivo (maximal plasma levels are 0.2 mM for adefovir and 2.0 mM for tenofovir), they do not inhibit metabolism of other drugs on the CYP level with an exception in a weak inhibition of CYP1A activity, that we have confirmed in the previous study. Unfortunately, there is no detailed information published on the methods used in the testing of the interactions of adefovir and tenofovir with cytochromes P450. In our previous study, we have shown that despite no general or large inhibition of the important microsomal CYPs, AD has inhibited CYP3A4 activity to some extent and both adefovir and tenofovir decreased metabolic activity of CYP2C9. Seeing the discrepancy to what has been published before, we have continued the studies on these antivirals on the gene expression level. The screening
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Figure 3. Western blotting for Cyp1a2, Cyp51 a Cyp26a1. M, marker; AD, adefovir dipivoxil; TD, tenofovir disoproxil; NK, negative control, PK, positive control; HLM, human liver microsomes.
phase of the study was aimed at finding cytochromes P450 differentially expressed after an in vivo application of the drugs tested. A microarray experiment was designed using an array specialized for a comprehensive analysis of the expression of cytochromes P450 metabolizing both endogenous and exogenous substrates and related genes, mainly from the cholesterol metabolism genes and CYP regulators. Specifically, Sterolgene Version 1 Mouse contains probes for 151 genes of the cholesterol metabolism including 71 murine CYP isoforms (there have been identified 103 CYP genes in mouse so far (Nelson, 2009)). The microarray experiment (28 hybridizations in total) then gave a complex overview of the drug effects and specified target genes for further detailed analysis by real-time PCR and immunoblotting. It has confirmed the functionality of the experimental design of the in vivo experiment, where the drugs administered to the animals to achieve a CYP induction have really caused it. These were two prototypic inducers of CYP2B10 and CYP1A2, phenobarbital a b-naphthoflavone, respectively. This biological settings approval is important due to mRNA levels variability and kinetics as too early or too late sampling can lead to false negative results when the mRNA was not synthesized yet or it has already accomplished its function and has been degraded, so there is no gene induction detectable on the mRNA level. Summarizing the results with the test inducers, we have observed a statistically and biologically significant induction of gene expression of Cyp1a2 (b-naphthoflavone inducible), Cyp2b10 (phenobarbital inducible), Cyp2b13, Cyp3a41, Cyp3a13, Cyp51a1, Cyp2a4, Cyp8b1 and Cyp2a12 (decreasing intensity). Besides cytochromes P450, a few other genes present on the Sterolgene array were induced. All these results were statistically significant at the level of 0.05. The Cyp1a2 induction has reached 12-folds and the induction of Cyp2b10 was 5-fold higher compared to control group which is a good level of gene expression change for an in vivo experiment. The results correspond to what has been
expected and confirm the sensitivity of the experimental system. On the other hand, there were hardly any genes significantly up- or down-regulated by AD or TD with the exception of Ebp (sterole-7,8-isomerase, emopamil binding protein) in TD group. This enzyme plays a role in cholesterol synthesis. Human CYP26A1 is not a typical xenobioticsmetabolizing enzyme. It is involved in regulation of retinoic acid cell signaling, which plays a role in fine control of differentiation and patterning of various stem/progenitor cell populations, also in embryo development. Both drugs have slightly impaired the expression of Cyp26a1 but the effect was only minor and cannot be considered biologically significant also because it was not supported by the real-time PCR result. CYP51 (lanosterol-140 -demethylase) is a family of highly conserved monooxygenases found in mycobacteria, fungi, plants, animals and humans. It metabolizes lanosterol to produce important downstream products as follicular fluidmeiosis activating steroid (FF-MAS) and testis-meiosis activating steroid (T-MAS) that affect ovarian and testicular function, and cholesterol, which is necessary for the synthesis of bile acids, mineralocorticoids, glucocorticoids and sex steroids. While cholesterol can, in principle, be supplemented with food intake, inhibition of CYP51 may affect the endocrine system. There was a mild down-regulation of Cyp51 and an induction of Cyp21a1 in the TD group. These changes were not further validated by PCR and we do not consider them biologically relevant except for Cyp51, where the expression level was 30% less than in control group as detected on microarray/47% less as detected by real-time PCR. If this can be considered to be a down-regulation of Cyp51, then it is a matter for discussion. Also the interpretation of Cyp26a1 down-regulation is difficult due to its small extent and a large inter-individual variability of the gene expression in the negative control group. Immunoblotting of the CYP26A1 protein was not successful as there is no
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anti-mouse CYP26A1 on the market and an anti-human immunoglobulin has no reactivity towards mouse CYP26A1. Real-time analysis has focused on genes playing role in metabolism of xenobiotics and the genes positive in the microarray experiment. It should provide a more detailed and specific data about the expression changes in the important genes. The results were in a great concordance with the microarray data even if the experimental design as well as statistical evaluation was completely different. Again, in the positive control group a large gene induction was observed for both the cytochromes inducible by phenobarbital or b-naphthoflavone, respectively, and the extent was comparable: 8.5-fold for Cyp1a2 and over 26-fold for Cyp2b10. A massive induction of CYP1A2 was seen on the western blot, where also specific degradation products were detected. For the mouse CYP2B10 protein, there was no antibody available. In the groups of animals treated with adefovir, dipivoxil or TD, the gene expression profile of the most important CYP isoforms was comparable to the negative control group. Interestingly, a mild down-regulation of the Cyp1a2 gene expression was observed for both drugs. It was confirmed on the protein level where an 2-fold decrease in signal was seen when comparing the AD group to negative control sample. Although there was a somewhat different overall intensity of individual immunoblots and an inter-individual variability in the negative control group, there was an obvious decrease of CYP1A2 protein in AD group. At the mRNA level, a 2-fold induction was seen for AD and Cyp2a4/5. Similar trends were observed in the TD group: a slight induction of the Cyp2a4 expression and a down-regulation of Cyp1a2, although the values have not reached the cut-off (the relative expression was 1.8 for Cyp2a4 and 0.68 for Cyp1a2). There was also a larger inter-individual variability in negative controls Cyp2a4 gene expression. The 2-fold down-regulation of the Cyp51 in the tenofovir group was observed only at the mRNA level which may be caused by a delay in protein synthesis. Other CYPs in the TD group as well as in AD group were showing a stable expression which made a contrast to positive control group, where all significantly changed cytochromes were up-regulated with the only exception of Cyp2e1 with a relative expression of 53% of the negative control group.
Conclusions Nucleoside analogues have some potential to interact with cytochromes P450. These drugs directly interact with DNA synthesis and repair and/or may affect different cell signalling pathways and processes in the cell and thus may directly or indirectly affect the drug metabolizing systems. Our previous study has shown an inhibition of activity of several CYP forms (especially CYP2C9) in human liver microsomes and recombinant human CYP enzymes by AD and TD. This work completes the knowledge with experimental results on their effects on CYP gene expression. Together they propose a general and universally usable scheme for testing drug interactions with cytochromes P450 as a major drugmetabolizing system. Such a thorough testing should be a base for clinical approval of each new drug.
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Gene expression changes in mice treated repeatedly with the drugs tested were only minor as detected on a Sterolgene microarray containing 40 CYP genes. The results were confirmed by qPCR and immunoblotting. Comparing the results of AD- and TD-treated animals to controls treated with prototypic CYP inducers it is obvious that the extent of the gene expression changes is small and their biological significance is probably low. Using a mouse model, our work indicates no clinically relevant risk of developing drug–drug interactions based on an induction of cytochrome P450 metabolism by AD or TD which is in fact favourable for these drugs and supports the evidence of safety of AD (Hepsera) and TD (Viread) with respect to drug–drug interactions based on enzyme induction.
Declaration of interest The work was supported by grants CZ.1.07/2.3.00/20.0019 of the Ministry of Education of the Czech Republic and European Social Funds (ESF) and CZ.1.05/2.1.00/01.003 – Biomedicine for regional development and human resources (BIOMEDREG).
References Anzenbacher P, Anzenbacherova´ E. (2001). Cytochromes P450 and metabolism of xenobiotics. Cell Mol Life Sci 58:737–47. Benhamou Y. (2006). Treatment algorithm for chronic hepatitis B in HIV-infected patients. J Hepatol 44:90–4. Curk T, Demsar J, Xu Q, et al. (2005). Microarray data mining with visual programming. Bioinformatics 21:396–8. De Clercq E, Holy A. (2005). Acyclic nucleoside phosphonates: a key class of antiviral drugs. Nat Rev Drug Discov 4:928–40. De Clercq E, Andrei G, Balzarini J, et al. (2005). Antiviral potential of a new generation of acyclic nucleoside phosphonates, the 6-[2(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines. Nucleosides Nucleotides Nucleic Acids 24:331–41. Delaney WE, Ray AS, Yang H, et al. (2006). Intracellular metabolism and in vitro activity of tenofovir against hepatitis B virus. Antimicrob Agents Chemother 50:2471–7. Demsar J, Zupan B, Leban G. (2004). Orange: from experimental machine learning to interactive data mining, White Paper, Faculty of Computer and Information Science, University of Ljubljana. Available at: http://www.ailab.si/orange [last accessed 14 Aug 2013]. FDA. (2011). Viral hepatitis therapies. Available at: http://www.fda. gov/ForConsumers/ByAudience/ForPatientAdvocates/ucm151494.htm [last accessed 21 Sept 2013]. Gnerre C, Schuster GU, Roth A, et al. (2005). LXR deficiency and cholesterol feeding affect the expression and phenobarbital-mediated induction of cytochromes P450 in mouse liver. J Lipid Res 46: 1633–42. Guengerich FP. (1995). Human cytochrome P450 enzymes. In: Ortiz de Montellano PR, ed. Cytochrome P450: structure, mechanisms and biochemistry. New York: Plenum, 473–535. Gutala RV, Reddy PH. (2004). The use of real-time PCR analysis in a gene expression study of Alzheimer’s disease post-mortem brains. J Neurosci Methods 132:101–7. Kearney BP, Flaherty JF, Shah J. (2004). Tenofovir disoproxil fumarate; clinical pharmacology and pharmacokinetics. Clin Pharmacokinet 43: 595–612. Kearney BP, Reul T, Coleman R, et al. (2000). Pharmacokinetics of adefovir dipivoxil (ADV) in combination with saquinavir (SQV), indinavir (IDV), efavirenz (EFV), delavirdine (DLV), didanosine (ddI), or lamivudine (3tc) in normal volunteers. Conf Retroviruses Opportunistic Infect 7:91 (abstract no 86). Nekvindova´ J, Masek V, Veinlichova´ A, et al. (2006). Inhibition of human liver microsomal cytochrome P450 activities by adefovir and tenofovir. Xenobiotica 36:1165–77.
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Nelson DR. (2009). The Cytochrome P450 Homepage. Human Genomics 4:59–65. Nunez M, Soriano V. (2005). Management of patients co-infected with hepatitis B virus and HIV. Lancet Infect Dis 5:374–82. Pfaffl MW. (2001). A new mathematical model for relative quantification real-time RT-PCR. Nucleic Acids Res 29:e45. Rezen T, Fon Tacer K, Kuzman D, et al. (2008). The Sterolgene v0 cDNA microarray: a systemic approach to studies of cholesterol homeostasis and drug metabolism. BMC Genomics, 9, 76. List of genes present on the Sterolgene v0 cDNA microarray. Available at: http://www.biomedcentral.com/content/supplementary/ 1471-2164-9-76-S1.pdf [last accessed 13 Sept 2013]. Rezen T, Tamasi V, Lo¨vgren-Sandblom A, et al. (2009). Effect of CAR activation on selected metabolic pathways in normal and hyperlipidemic mouse livers. BMC Genomics 10:384. WHO. (2013a). Consolidated guidelines on general HIV care and the use of antiretroviral drugs for treating and preventing HIV infection: recommendations for a public health approach. 1. HIV
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infections – drug therapy. 2. HIV infections – prevention and control. 3. Anti-Retroviral agents – therapeutic use. 4. Guideline. ISBN 978 92 4 150572 7. Available at: http://apps.who.int/iris/ bitstream/10665/85321/1/9789241505727_eng.pdf [last accessed 19 Sept 2013]. WHO. (2013b). HIV/AIDS. Fact sheet N 360. Updated October 2013. Available at: http://www.who.int/mediacentre/factsheets/fs360/en/ index.html [last accessed 9 Nov 2013]. WHO. (2013c). Hepatitis B. Fact sheet N 204. Updated July 2013. Available at: http://www.who.int/mediacentre/factsheets/fs204/en/ index.html [last accessed 19 Sept 2013]. Yang D, Wu L, Hwang YS, et al. (2001). Expression of the REB transcriptional activator in rice grains improves the yield of recombinant proteins whose genes are controlled by a Reb-responsive promoter. Proc Natl Acad Sci USA 98:11438–43. Zhang WX, Lai V, Mutimer D, Mirza D. (2006). Adefovir dipivoxil for the treatment of lamivudine-resistant hepatitis B mutants. Hepatobiliary Pancreat Dis Int 5:154–6.
RESEARCH ARTICLE
Acyclic nucleoside phosphonates: a study on cytochrome P450 gene expression Jana Nekvindova1, Juan Antonio Contreras2, Peter Juvan2,3, Klementina Fon Tacer2,4, Pavel Anzenbacher1, Zdenek Zidek5, Michaela Kopecna Zapletalova1, Damjana Rozman2, and Eva Anzenbacherova1 1
Faculty of Medicine and Dentistry, Institute for Molecular and Translational Medicine, Palacky University, Olomouc, Czech Republic, Faculty of Medicine, Centre for Functional Genomics and Biochips, Institute of Biochemistry, Ljubljana, Slovenia, 3Artificial Intelligence Laboratory, Faculty of Computer and Information Science, University of Ljubljana, Ljubljana, Slovenia, 4Physiology Department, Southwestern Dallas Medical Center, University of Texas, Dallas, TX, USA, and 5Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
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2
Abstract
Keywords
1. Nucleotide analogues comprise an important class of drugs used in treatment of viral infections but also cancer. These drugs affect the structural integrity of DNA and activate different pathways and processes in the cell and may directly or indirectly influence the drug metabolizing system. Adefovir dipivoxil (AD) and tenofovir disoproxil (TD) are nucleotide analogues approved for the treatment of chronic hepatitis B and/or HIV/AIDS infection. 2. To evaluate the risk of their drug–drug interactions on the level of drug metabolism, an effect of both compounds on cytochromes P450 expression was studied using cDNA microarrays, real-time RT-PCR and immunoblotting. Mice were given intraperitoneally 25 mg/kg of AD or TD, respectively. As a positive control, a combination of prototypic cytochromes P450 (CYP) inducers, phenobarbital and b-naphthoflavone was chosen. 3. The data obtained showed a significant CYP induction in the positive control group, but no clinically significant induction of CYP genes by AD or TD was observed. Our results support the evidence of safety of AD and TD with respect to drug–drug interactions based on enzyme induction. These findings are important as a plethora of new antivirals of different types are being tested and introduced to clinical practice, mostly to be used in combinations.
Adefovir, antiviral, CYP, drug metabolism, induction, PMEA, PMPA, tenofovir
Introduction Drug side effects or toxicity as well as failure of therapy are often caused by drug–drug interactions on the level of enzymes of drug metabolism. Here, enzyme induction or inhibition leading to changes in metabolic activity of an enzyme may result in changed levels of the drugs metabolized. In particular cases, this includes also an under dosing of drugs that need to be enzymatically activated due to enzyme deficiency or, on the other hand, an ineffective detoxication and elimination of drugs due to inhibition of enzymes responsible for their inactivation. Most of the drug interactions are rather unpredictable without a systematic experimental and clinical testing. Especially for drugs that directly interact with DNA synthesis and reparation and/or
Address for correspondence: Jana Nekvindova, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 5, 779 00 Olomouc, Czech Republic. Tel: +420 495 832 767. E-mail: [email protected] Eva Anzenbacherova, Institute of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic. Tel: +420 585 632 321. E-mail: [email protected]
History Received 8 January 2014 Revised 13 February 2014 Accepted 14 February 2014 Published online 5 March 2014
may affect cell cycle and/or different cell signalling pathways, an effect on transcription of various genes should be carefully evaluated. A solid base for such an evaluation of a particular drug in terms of its drug interactions is provided by in vitro and in vivo testing of the drug’s effect on the cytochrome P450 system. Cytochromes P450 (CYP, pl. CYPs) are the most important drug metabolizing enzymes in man (Anzenbacher & Anzenbacherova´, 2001; Guengerich, 1995). They also participate in synthesis of important endogenous compounds, e.g. steroid hormones, prostaglandins and thromboxanes (Anzenbacher & Anzenbacherova´, 2001). Regulation of their expression and activity is a multi-level complex process that is despite all current knowledge in this field not completely understood. Acyclic nucleoside phosphonates are modern antivirals with a potent activity against many DNA viruses and retroviruses. Their value consists in a broader spectrum of activity, a longer duration of their effect and a lower risk of emerging viral resistance to the therapy (De Clercq & Holy, 2005). Chemical structure of the drugs is depicted in Figure 1. Adefovir dipivoxil (AD) (bis(pivaloyloxymethyl)ester of 9-[(2-phosphonomethoxy) ethyl]adenine) is a nucleoside
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Figure 1. Structure of the compounds studied.
analogue approved for treatment of chronic hepatitis B, where only seven drugs are currently available: entecavir (Baraclude), lamivudine (Epivir-HBV), AD (Hepsera), interferon alpha-2b (Intron A), pegylated interferon (Pegasys), telbivudine (Tyzeka) and tenofovir (Viread) (FDA, 2011). It can be used also for the treatment of lamivudine-resistant HBV mutants (Benhamou, 2006; Nunez & Soriano, 2005; Zhang et al., 2006). For hepatitis B suppression, AD is given at a dose of 10 mg daily. Higher doses (60 or 120 mg/day) suppress also HIV viral replication but these are nephrotoxic in man. Tenofovir disoproxil (TD) (bis-isopropyloxycarbonyloxymethyl ester of 9-R-[2-(phosphonomethoxy)propyl]adenine) is used – as its fumarate (tenofovir DF, VireadÕ ) – in antiretroviral therapy (ART) of an HIV/AIDS infection; currently, it is approved also for the treatment of chronic hepatitis B (Delaney et al., 2006; FDA, 2011). For HIV infection, tenofovir DF is included in first line therapeutic regimen according to the June 2013 version of the consolidated guidelines on general HIV care and the use of antiretroviral drugs for treating and preventing HIV infection (WHO, 2013a). Recently, several combinations of tenofovir DF with other antivirals have been approved by FDA as a single preparation: Truvada (tenofovir DF + emtricitabine), Atripla (tenofovir DF + emtricitabine +efavirenz), Complera (emtricitabine + rilpivirine hydrochloride + tenofovir DF) and four combination Stribild (cobicistat + elvitegravir + emtricitabine + tenofovir DF) but more and new combinations are clinically tested and standardized. The need for safe drugs becomes more important both medically and socioeconomically with the rapidly increasing number of drugs introduced to the market and improving of the peoples’ access to pharmacotherapy. According to the World Health Organization (WHO) data, there were 35.3 million people suffering from HIV/AIDS infection. More than 9.7 million were receiving ART in 2012. Of this, 630 000 were children. This is over 30-fold increase in 10 years and close to a 20% increase in just 1 year (WHO, 2013b) For hepatitis B, there are 240 million of chronic cases and 600 000 people are estimated to die every year because of HBV infection (WHO, 2013c). Since both adefovir and tenofovir are highly important in the treatment of HBV and HIV infections, as well as they are used in combinations, the studies aiming at evaluation of their potential of drug– drug interactions are of particular interest. According to prescribing information, tenofovir DF interacts with didanosine (mechanism unknown) and HIV-1 protease inhibitors,
e.g. atazanavir and ritonavir (increased absorption of tenofovir DF). Concerning the possibility of drug interactions on the level of cytochromes P450, an inhibition of activities several human microsomal CYP forms by AD, TD and their active forms, adefovir and tenofovir, has been observed (Nekvindova´ et al., 2006). The most significant inhibition occurred in the case of CYP2C9 and CYP3A4; tenofovir and TD inhibited also CYP2E1 and CYP1A2 activities. These findings were in contrast to previously published data stating that the drugs are not substrates, inhibitors or inducers of CYPs (Kearney et al., 2000, 2004). However, as the inhibition occurred in rather high concentrations of these drugs, the inhibition of CYP activities is most probably not clinically significant. This sequential study is focused on evaluation of another mechanism of drug interactions, namely, on the potential of AD and TD to induce cytochromes P450 which has not been evaluated to date.
Materials and methods Animals and RNA preparation Animal experiments were evaluated and approved by Ethical committee of the Palacky University, Faculty of Medicine and Dentistry. Female mice of the inbred strain C57BL/6, 8–10 weeks old, were purchased from Charles River Deutschland (Sulzfeld, Germany). They were kept in transparent plastic cages and maintained in an Independent Environmental Air Flow Animal Cabinet (ESI Flufrance, Wissous, France). Lighting was set from 6 AM to 6 PM, temperature set at 22 C. Animals were randomly separated into four groups and treated for three consecutive days by intraperitoneal injections as following: AD and TD groups (five mice in each group) were given 25 mg/kg of AD or TD, respectively. PC (positive control) group of four animals was treated with 60 mg/kg phenobarbital plus one dose of 80 mg/kg of b-naphthoflavone on day two. Five animals of NC (negative control) group were injected 200 ml of 0.9% saline solution. On day four, animals were killed by cervical dislocation. Total RNA was isolated from liver tissue samples stored in RNAlater (Qiagen, Hilden, Germany) using TRI REAGENT (MRC, Cincinnati, OH). RNA concentration and purity was measured by spectrophotometry at 260, 280 and 230 nm using an Infinite M200 micro plate reader (Tecan, Ma¨nnedorf, Switzerland). RNA integrity was determined on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano LabChip kit (both Agilent Technologies, Santa Clara, CA).
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Table 1. Primer sequences for real-time PCR. Cyp1a2 Cyp2a4 Cyp2d9 Cyp2e1 Cyp3a13 Cyp8b1 Cyp2b10 Cyp51 LDLr 18S rRNA
50 -GCACTACCAAGACTTCAACAAGAACA-30 50 -AGGTACAGAAGAAACAGAGGACACTTC-30 50 -CCAACCCCATGCTGAACAAATCCAC-30 50 -GTTCCAGGAGTACAAGAACAAGGGG-30 50 -TGGGGACGATTCTTGCTTACCAGAA-30 50 -AAGGCTGGCTTCCTGAGCTT-30 50 -CAATGTTTAGTGGAGGAACTGCG-30 50 -ACGCTGCCTGGCTATTGC-30 50 -AGGCTGTGGGCTCCATAGG-30 50 -CGCCGCTAGAGGTGAAATTC-30
50 -TGTGGTGACTGTGTCAAAGCCAG-30 50 -TATTATTCCTATTGACAACATAGTATAATTTCCCC-30 50 -GGATACGCAAGAGTATCGGGAATGC-30 50 -CCATTCCCCAGTCACGGAGGAT-30 50 -GCTGTCGACCGTCATACAACCCC-30 50 -AACAGCTCATCGGCCTCATC-30 50 -CACTGGAAGAGGAACGTGGG-30 50 -TTGATCTCTCGATGGGCTCTATC-30 50 -TGCGGTCCAGGGTCATCT-30 50 -TTGGCAAATGCTTTCGCTC-30
Gnerre et al. (2005); Yang et al. (2001) (Cyp51, LDLr); Gutala & Reddy (2004) (18S rRNA).
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Microarray hybridization cDNA microarrays were developed in Centre for Functional Genomics and Bio-chips (CFGBC) of the University of Ljubljana. The arrays contained probes for 151 genes involved in cholesterol metabolism including genes of 40 cytochrome P450 forms (Cyp1a1, 1a2, 1b1, 2a4, 2a12, 2b9, 2b10, 2b13, 2c40, 2d22, 2e1, 2f2, 2g1, 2j5, 2j6, 3a11, 3a13, 3a25, 3a41, 4b1, 4f14, 5a1, 7a1, 7b1, 8a1, 8b1, 11a1, 11b2, 17a1, 19a1, 20a1, 21a1, 24a1, 26a1, 26b1, 27a1, 27b1, 39a1, 46a1 and 51a1). The hybridization protocol was similar to previously published (Rezen et al., 2008, 2009). Briefly, indirect amino-allyl (aa-) labelling and hybridization protocol was used. cDNA synthesis and incorporation of aa-dUTP by reverse transcription was performed using SuperScriptTM III Reverse Transcriptase (Invitrogen, Carlsbad, CA) with oligo dT primers. Luciferase Control RNA (Promega, Fitchburg, WI) and Lucidea Universal ScoreCardTM RNA (test and reference RNA mixes) were used as internal controls and calibrators. Cy3 and Cy5 NHS esters were coupled to aacDNA, purified samples were hybridized overnight (16 h) on the microarrays. When hybridization was done, array slides were washed and scanned with Tecan LS200 Scanner (Tecan, Ma¨nnedorf, Switzerland). Fluorescence images were obtained and analyzed using Array-Pro Analyzer software (Media Cybernetics, Rockville, MD). Data normalization and analysis were performed using Orange (Curk et al., 2005; Demsar et al., 2004) software. Direct comparison design was used in all microarray experiments. All the samples were hybridized against pooled negative control samples and a dye-swap was used to produce two technical replicates. Statistical significance evaluation of the normalized Cy3 and Cy5 fluorescence was done by Student’s t-test at levels 0.05 and 0.1 for a more complex evaluation. Real-time RT-PCR First strand cDNAs were synthesized from total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers (Promega). RNA was pre-treated with DNase I (Sigma-Aldrich, St. Louis, MO) and protected with RNase OUT (Invitrogen). Real-time PCR was performed on ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Target genes were amplified using the following set of primers listed in Table 1. 18S rRNA was used as an internal control for other genes data normalization. Optimized PCR reactions were performed in 20 ml volume in 96well plate format, using hot-start technology Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) with ROX
reference dye. Carry-over contamination was controlled by using non-template control samples. Thermal profile of the cycling program was: 50 C for 2 min hold (UDG incubation), 95 C for 2 min hold (Taq polymerase denaturation/activation) and up to 40 cycles of 95 C for 15 s and 60 C for 60 s). Data analysis was performed on ABI 7900HT software and statistic evaluation was done in Microsoft Office Excel 2003 using ddCT calculation method (Pfaffl, 2001) comparing each sample from drug-treated animals to each of the negative control animals (Table 1).
Results First, the microarray experiment was performed to get a global view over expression changes. Then a set of genes was chosen for a detailed examination by real-time RT-PCR and where a specific antibody was available, immunoblotting was done to confirm the results on the protein level. The data are presented according to this sequence and individual groups treated by different drugs are compared. All the results obtained were in concordance and gave a confident idea about the CYP inductions by the drugs used in the experiment. Microarray experiment Raw microarray data and experimental design details have been deposited to Gene Expression Omnibus database (GEO, http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE47698. Samples from drug-treated animals were hybridized one by one against pooled negative control samples. Statistical analysis of normalized Cy5 and Cy3 fluorescence signals of individual probes on the array revealed a set of genes differentially expressed in all groups of drug-treated animals when compared to negative controls treated by vehiculum alone. The outcoming set of genes was the largest in the positive control group (phenobarbital plus b-naphthoflavone treated), fewer genes were differentially expressed in animals treated by TD and only minor changes were detected in animals treated by AD (Table 1). Log2 ratio was evaluated and a biological cut off was set to ±0.9 (somewhat 52-fold up- or down-regulation) according to statistical significance, experimental design and mapping of known effects of CYP inducers. Major changes were detected in the positive control (PC) group and target genes, which were Cyp1a2 for b-naphthoflavone and Cyp2b10 for phenobarbital. In this PC group, an up-regulation of 12-fold (log2 ratio 3.584) was observed for Cyp1a2 and nearly 5-fold (log2 ratio 4.8) for Cyp2b10 as it is depicted in Table 2.
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Table 2. Differentially expressed genes in control- and drug-treated groups, statistically significant changes at p ¼ 0.05.
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Gene symbol
Gene name
Positive control (phenobarbital plus b-naphtoflavone) Cyp1a2 Cytochrome P450 1a2 Cyp2b10 Cytochrome P450 2b10 Cyp2b13 Cytochrome P450 2b13 Abcc3 ATP-binding cassette, sub-family C (CFTR/MRP), member 3 Cyp3a41 Cytochrome P450 3a41 Cyp3a13 Cytochrome P450 3a13 Cyp51a1 Cytochrome P450 51a1 Dhcr24 24-Dehydrocholesterol reductase Cyp2a4 Cytochrome P450 2a4 Nsdhl AD(P) dependent steroid dehydrogenase-like Lss Lanosterol synthase Fdps Farnesyl diphosphate synthetase Actb2 Actin, beta Cyp8b1 Cytochrome P450 8b1 Cyp2a12 Cytochrome P450 2a12 Lip1 Lysosomal acid lipase A Alas1 Aminolevulinic acid synthase 1 Ppia Peptidylprolyl isomerase A Acss2 Acyl-CoA synthetase short-chain family member 2 Apoa1 Apolipoprotein A-I Cyp2c40 Cytochrome P450 2c40 Cyp2f2 Cytochrome P450 2f2 Abcc2 ATP-binding cassette, sub-family C (CFTR/MRP), member 2 Ppara Peroxisome proliferator activated receptor alpha Cyp3a25 Cytochrome P450 3a25 Slc10a1 Solute carrier family 10 (sodium/bile acid cotransporter family), member 1 Nr1d2 Nuclear receptor subfamily 1, group D, member 2 Adefovir Scap SREBP cleavage activating protein (SREBP chaperone) Cyp26a1 Cytochrome P450 26a1 Tenofovir Ebp Phenylalkylamine Ca2+ antagonist (emopamil) binding protein Scarb1 Scavenger receptor class B, member 1 Acss2 Acyl-CoA synthetase short-chain family member 2 Cyp26a1 Cytochrome P450 26a1 Rxrb Retinoid X receptor beta Abcb4 ATP-binding cassette, sub-family B, member 4 (P-gp 2, MDR2) Gapd2 Glyceraldehyde-3-phosphate dehydrogenase Sc4mol Sterol-C4-methyl oxidase-like Ldlr Low-density lipoprotein receptor Ncor1 Nuclear receptor co-repressor 1, RXR interacting protein 13 Nr2b1 Retinoid X receptor alpha Cyp21a1 Cytochrome P450 21a1 Cyp51a1 Cytochrome P450 51a1 Mvk Mevalonate kinase Hmgcs1 3-Hydroxy-3-methylglutaryl-Coenzyme A synthase 1 Dhcr7 7-Dehydrocholesterol reductase
Log2 ratio
Fold change
3.584 2.258 1.881 1.836 1.641 1.585 1.336 1.273 1.211 1.205 1.135* 1.057 0.996 0.952 0.924 0.919 0.865 0.792 0.79 0.786 0.689 0.622 0.574 0.166 0.675* 0.418* 0.293*
12.0 4.8 3.7 3.6 3.1 3.0 2.5 2.4 2.3 2.3 2.2 2.1 2.0 1.9 1.9 1.9 0.5 1.7 0.6 1.7 0.6 0.6 1.5 0.9 1.6 0.7 0.8
0.376 0.536*
1.3 0.7
0.952 0.845 0.844 0.838 0.827 0.815 0.763 0.748 0.568 0.528 0.304 0.728* 0.569* 0.494* 0.414* 0.346*
1.9 1.8 0.6 0.6 1.8 1.8 1.7 0.6 1.5 1.4 1.2 1.7 0.7 1.4 1.3 1.3
Biological significance cut-off was set to an expression change of log2ratio ±0.9 (2-fold up- or down-regulation). Other statistically significant differences are shown for better illustration. *p ¼ 0.1.
Real-time RT-PCR The microarray results were confirmed by quantitative PCR where appropriate and a high concordance of both methods was seen. Genes with a suggested key role in drug metabolism in mice and genes that showed significant changes in the microarray experiment were selected for real-time PCR assays, namely Cyp1a2, Cyp2a4, Cyp2d9, Cyp2e1, Cyp3a13, Cyp8b1, Cyp2b10, Cyp51 and LDLr. Relative expression (RE) of a gene of interest was calculated by ddCT method against a selected reference gene (18S rRNA), always as an average of three technical replicates. At first, RE values (RE) ¼ 2 ddCT of all drugtreated samples compared to negative control samples were expressed by the logic ‘‘each to each’’, then an average value
for a particular sample (drug-treated animal) was calculated and finally an average RE with its standard deviation and standard error of the mean was obtained for the individual groups (AD, TD, PC, NC) (Table 3). Comparison of the positive results, where a gene expression change was detected by the microarray as well as by realtime PCR is necessary as a validation of the microarray results. Similar trends as well as value ranges were observed for most of the results, where a difference was seen the realtime results is considered to be correct as it is more specific, sensitive and accurate. Nevertheless, all but one significant gene expression changes were confirmed. The only exception was LDLr gene in TD group, where the discrepancy was related to the very low RE change (log2 ratio was close to 0), in other words, the gene expression was almost the same
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Table 3. Relative expression of selected genes – real-time PCR results. AD
TD
PC
NC
Gene
RE
SD
SEM
RE
SD
SEM
RE
SD
SEM
RE
SD
SEM
Cyp1a2 Cyp2a4 Cyp2d9 Cyp2e1 Cyp3a13 Cyp8b1 Cyp2b10 Cyp51 LDLr Cyp26a1
0.49 2.02 0.72 0.67 0.8 1.31 0.75 0.94 0.78 1.02
0.17 1.57 0.51 0.22 0.16 1.11 0.24 0.59 0.21 1.19
0.13 1.1 0.42 0.19 0.14 0.79 0.19 0.51 0.17 1.76
0.68 1.81 0.81 0.84 0.88 0.68 0.72 0.53 0.78 0.75
0.21 1.42 0.4 0.26 0.22 0.57 0.22 0.26 0.25 1.18
0.16 0.99 0.34 0.22 0.18 0.43 0.17 0.2 0.21 0.85
8.53 1.63 2.47 0.54 2.09 1.58 26.36 2.18 0.72 1.32
2.8 1.38 1.03 0.27 0.48 0.72 9.72 0.92 0.24 1.97
2.23 0.98 0.86 0.24 0.38 0.56 7.77 0.74 0.19 1.48
1.05 1.37 1.24 1.12 1.02 1.16 1.04 1.04 1.03 4.83
0.32 1.21 0.87 0.55 0.22 0.66 0.28 0.28 0.26 9.87
0.25 0.85 0.63 0.41 0.18 0.5 0.22 0.22 0.18 6.11
AD, adefovir dipivoxil; TD, tenofovir disoproxil; PC, positive control (phenobarbital + b-naphthoflavone); NC, negative control group; RE, relative expression (average fold induction, 1.00 ¼ 100% RENC); SD, standard deviation; SEM, standard error of the mean. Gene expression variability of the target genes in the negative control group is shown for illustration.
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was limited and where the antibody was targeted against human isoform, poor cross-reactivity with murine substrates led to an ineffective binding of the antibody. Reproducible results were obtained for CYP1A2 where a massive enzyme induction was detected in the positive control group and even specific degradation products of this protein were present on the blot (Figure 3). Samples in other groups were similar to each other as well as to negative control samples, i.e. there was no significant Cyp1a2 gene expression change on the protein level in AD or TD group. Incubation of the membranes with an anti-CYP51 antibody gave repeatedly similar results even if the reaction was not optimal and the signal intensity always varied between individual membranes. Using internal standards (negative/positive group samples), the comparison was possible. The anti-CYP26A1 was antihuman and despite all optimization effort it never reacted to murine samples (positive reaction is seen only for human liver microsomes, Figure 3).
Discussion
Figure 2. A comparison of the relative expression results obtained by cDNA microarrays Sterolgene v.1 Mouse and real-time RT-PCR. TD, tenofovir disoproxil-treated group; PC, positive control group; RE, relative expression.
in drug-treated animals as in negative controls. Expression of other genes was confirmed by real-time PCR even despite a different experimental approach and a completely different statistical analysis which increases the overall confidence of the results. The genes with highest correlation were Cyp1a2 and Cyp2b10 (induced by phenobarbital and b-naphthoflavone), Cyp2a4, 3a13, 8b1 and Cyp51 in the positive control group and Cyp51 gene (down-regulation) in the TD group as depicted in Figure 2. Western blotting was used to visualize gene induction on the protein level. The spectrum of antibodies on the market
Adefovir and tenofovir are potent antivirals successfully used in pharmacotherapy of chronic hepatitis B and HIV/AIDS infection, respectively. They are active against a broad spectrum of DNA viruses, e.g. polyoma-, papilloma-, adeno-, herpes- and poxviruses (De Clercq et al., 2005). The producer states on the drug’s web sites (http:// www.hepsera.com, http://www.viread.com) that these drugs are not substrates of CYPs and even in 4000 (adefovir) or 300 (tenofovir) higher concentrations than those reached in vivo (maximal plasma levels are 0.2 mM for adefovir and 2.0 mM for tenofovir), they do not inhibit metabolism of other drugs on the CYP level with an exception in a weak inhibition of CYP1A activity, that we have confirmed in the previous study. Unfortunately, there is no detailed information published on the methods used in the testing of the interactions of adefovir and tenofovir with cytochromes P450. In our previous study, we have shown that despite no general or large inhibition of the important microsomal CYPs, AD has inhibited CYP3A4 activity to some extent and both adefovir and tenofovir decreased metabolic activity of CYP2C9. Seeing the discrepancy to what has been published before, we have continued the studies on these antivirals on the gene expression level. The screening
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Figure 3. Western blotting for Cyp1a2, Cyp51 a Cyp26a1. M, marker; AD, adefovir dipivoxil; TD, tenofovir disoproxil; NK, negative control, PK, positive control; HLM, human liver microsomes.
phase of the study was aimed at finding cytochromes P450 differentially expressed after an in vivo application of the drugs tested. A microarray experiment was designed using an array specialized for a comprehensive analysis of the expression of cytochromes P450 metabolizing both endogenous and exogenous substrates and related genes, mainly from the cholesterol metabolism genes and CYP regulators. Specifically, Sterolgene Version 1 Mouse contains probes for 151 genes of the cholesterol metabolism including 71 murine CYP isoforms (there have been identified 103 CYP genes in mouse so far (Nelson, 2009)). The microarray experiment (28 hybridizations in total) then gave a complex overview of the drug effects and specified target genes for further detailed analysis by real-time PCR and immunoblotting. It has confirmed the functionality of the experimental design of the in vivo experiment, where the drugs administered to the animals to achieve a CYP induction have really caused it. These were two prototypic inducers of CYP2B10 and CYP1A2, phenobarbital a b-naphthoflavone, respectively. This biological settings approval is important due to mRNA levels variability and kinetics as too early or too late sampling can lead to false negative results when the mRNA was not synthesized yet or it has already accomplished its function and has been degraded, so there is no gene induction detectable on the mRNA level. Summarizing the results with the test inducers, we have observed a statistically and biologically significant induction of gene expression of Cyp1a2 (b-naphthoflavone inducible), Cyp2b10 (phenobarbital inducible), Cyp2b13, Cyp3a41, Cyp3a13, Cyp51a1, Cyp2a4, Cyp8b1 and Cyp2a12 (decreasing intensity). Besides cytochromes P450, a few other genes present on the Sterolgene array were induced. All these results were statistically significant at the level of 0.05. The Cyp1a2 induction has reached 12-folds and the induction of Cyp2b10 was 5-fold higher compared to control group which is a good level of gene expression change for an in vivo experiment. The results correspond to what has been
expected and confirm the sensitivity of the experimental system. On the other hand, there were hardly any genes significantly up- or down-regulated by AD or TD with the exception of Ebp (sterole-7,8-isomerase, emopamil binding protein) in TD group. This enzyme plays a role in cholesterol synthesis. Human CYP26A1 is not a typical xenobioticsmetabolizing enzyme. It is involved in regulation of retinoic acid cell signaling, which plays a role in fine control of differentiation and patterning of various stem/progenitor cell populations, also in embryo development. Both drugs have slightly impaired the expression of Cyp26a1 but the effect was only minor and cannot be considered biologically significant also because it was not supported by the real-time PCR result. CYP51 (lanosterol-140 -demethylase) is a family of highly conserved monooxygenases found in mycobacteria, fungi, plants, animals and humans. It metabolizes lanosterol to produce important downstream products as follicular fluidmeiosis activating steroid (FF-MAS) and testis-meiosis activating steroid (T-MAS) that affect ovarian and testicular function, and cholesterol, which is necessary for the synthesis of bile acids, mineralocorticoids, glucocorticoids and sex steroids. While cholesterol can, in principle, be supplemented with food intake, inhibition of CYP51 may affect the endocrine system. There was a mild down-regulation of Cyp51 and an induction of Cyp21a1 in the TD group. These changes were not further validated by PCR and we do not consider them biologically relevant except for Cyp51, where the expression level was 30% less than in control group as detected on microarray/47% less as detected by real-time PCR. If this can be considered to be a down-regulation of Cyp51, then it is a matter for discussion. Also the interpretation of Cyp26a1 down-regulation is difficult due to its small extent and a large inter-individual variability of the gene expression in the negative control group. Immunoblotting of the CYP26A1 protein was not successful as there is no
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anti-mouse CYP26A1 on the market and an anti-human immunoglobulin has no reactivity towards mouse CYP26A1. Real-time analysis has focused on genes playing role in metabolism of xenobiotics and the genes positive in the microarray experiment. It should provide a more detailed and specific data about the expression changes in the important genes. The results were in a great concordance with the microarray data even if the experimental design as well as statistical evaluation was completely different. Again, in the positive control group a large gene induction was observed for both the cytochromes inducible by phenobarbital or b-naphthoflavone, respectively, and the extent was comparable: 8.5-fold for Cyp1a2 and over 26-fold for Cyp2b10. A massive induction of CYP1A2 was seen on the western blot, where also specific degradation products were detected. For the mouse CYP2B10 protein, there was no antibody available. In the groups of animals treated with adefovir, dipivoxil or TD, the gene expression profile of the most important CYP isoforms was comparable to the negative control group. Interestingly, a mild down-regulation of the Cyp1a2 gene expression was observed for both drugs. It was confirmed on the protein level where an 2-fold decrease in signal was seen when comparing the AD group to negative control sample. Although there was a somewhat different overall intensity of individual immunoblots and an inter-individual variability in the negative control group, there was an obvious decrease of CYP1A2 protein in AD group. At the mRNA level, a 2-fold induction was seen for AD and Cyp2a4/5. Similar trends were observed in the TD group: a slight induction of the Cyp2a4 expression and a down-regulation of Cyp1a2, although the values have not reached the cut-off (the relative expression was 1.8 for Cyp2a4 and 0.68 for Cyp1a2). There was also a larger inter-individual variability in negative controls Cyp2a4 gene expression. The 2-fold down-regulation of the Cyp51 in the tenofovir group was observed only at the mRNA level which may be caused by a delay in protein synthesis. Other CYPs in the TD group as well as in AD group were showing a stable expression which made a contrast to positive control group, where all significantly changed cytochromes were up-regulated with the only exception of Cyp2e1 with a relative expression of 53% of the negative control group.
Conclusions Nucleoside analogues have some potential to interact with cytochromes P450. These drugs directly interact with DNA synthesis and repair and/or may affect different cell signalling pathways and processes in the cell and thus may directly or indirectly affect the drug metabolizing systems. Our previous study has shown an inhibition of activity of several CYP forms (especially CYP2C9) in human liver microsomes and recombinant human CYP enzymes by AD and TD. This work completes the knowledge with experimental results on their effects on CYP gene expression. Together they propose a general and universally usable scheme for testing drug interactions with cytochromes P450 as a major drugmetabolizing system. Such a thorough testing should be a base for clinical approval of each new drug.
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Gene expression changes in mice treated repeatedly with the drugs tested were only minor as detected on a Sterolgene microarray containing 40 CYP genes. The results were confirmed by qPCR and immunoblotting. Comparing the results of AD- and TD-treated animals to controls treated with prototypic CYP inducers it is obvious that the extent of the gene expression changes is small and their biological significance is probably low. Using a mouse model, our work indicates no clinically relevant risk of developing drug–drug interactions based on an induction of cytochrome P450 metabolism by AD or TD which is in fact favourable for these drugs and supports the evidence of safety of AD (Hepsera) and TD (Viread) with respect to drug–drug interactions based on enzyme induction.
Declaration of interest The work was supported by grants CZ.1.07/2.3.00/20.0019 of the Ministry of Education of the Czech Republic and European Social Funds (ESF) and CZ.1.05/2.1.00/01.003 – Biomedicine for regional development and human resources (BIOMEDREG).
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